The present invention relates to novel DNA constructs comprising a vector linked to a DNA segment which encodes a protein containing a signal protein at its N-terminus and an anchor sequence at its C-terminus.
More particularly, the present invention relates to vaccines which are useful for the prevention and treatment of malaria caused by Plasmodium falciparum in humans.
This work was supported by a DARPA grant. The government retains certain rights in the invention.
Preventing or treating malaria has long been a challenging health problem, particularly in developing countries, and the rapid development of drug resistance in the parasite has enhanced the need for the development of a malaria vaccine. Although there has been steady progress over the last decade, several problems still must be overcome, including selection of an appropriate delivery system vehicle and antigen carrier.
Although malaria was believed to have subsided after World War II, recent outbreaks suggest that the disease is on the rise. Malaria is again the leading cause of morbidity/mortality globally and presents an increasing threat in at risk environments. Estimates are that 300 million new cases of malaria occur each year, with mortality of approximately 1% of infected individuals. Prophylactic medications used to prevent the disease have been rendered ineffective by the emergence of drug-resistant strains of the parasite worldwide. Complete vector protection is simply not possible and all attempts to eradicate the relevant species of mosquito have failed.
Four species of protozoa of the genus Plasmodium are found in man. The four species include: Plasmodium vivax, Plasmodium malariae, Plasmodium falciparum and Plasmodium ovale. Of these, Plasmodium falciparum produces the most pathogenic of the malarias and often results in death. It is responsible for about half of the human cases of malaria found worldwide.
In malaria, the disease is such that infection followed by recovery does not confer meaningful protection to the individual despite a significant antibody response to several of the parasite proteins. The conventional wisdom has been that the parasite either does not possess antigens suitable for the development of a protective response or has evolved mechanisms which allow it to escape the host immune system. Because recent evidence has shown that immune protection is possible using irradiated sporozoites, the latter hypothesis described above is the more reasonable explanation.
The life cycle of the malaria parasite provides several stages at which interference could lead to cessation of the infective process. Included among these stages is the invasion of the erythrocyte by the merozoite. The merozoite represents a potentially attractive target (and perhaps the only target) from which a vaccine may be produced, because the free merozoite, although it has a limited lifetime (one to two hours) occurs earlier in the life cycle of malaria, and the emergence of later stage sexual forms, which are responsible for transmission of the disease, depends upon the erythrocytic stage.
The general life cycle of malaria parasites is the same for human and other animal malaria parasites, thus allowing model studies to be conducted on a rodent species with accurate translation to the human parasite. For example, the rodent malaria parasite strain Plasmodium berghei Anka has a pathology very similar to the FCR-3 strain of Plasmodium falciparum (a well studied variant of the human parasite). In addition, the blood stage of the human parasite can be grown in the laboratory (in human red cells) thus affording a system for studying the effects of antibodies/inhibitors on the invasion process, and the erythrocytic phase.
In the life cycle of the malaria parasite, a human becomes infected with malaria from the bite of a female Anopheles mosquito. The mosquito inserts its probe into a host and in so doing, injects a sporozoite form of Plasmodium falciparum, present in the saliva of the mosquito.
The sporozoites which have been injected into the human host are cleared into a number of host tissue cells, including liver parenchyma cells (hepatocytes) and macrophages. This phase is known as the exoerythrocytic cycle because at this point in the life cycle the organism has not yet entered red blood cells. After entering hepatocytes, sporozoites undergo a transformation into trophozoites, which incubate and undergo schizogony, rupture and liberate tissue merozoites. This process takes approximately 7-10 days and, depending upon species, may repeat itself several times, during which time the host feels no effects. In Plasmodium falciparum, this repetition does not occur. After the incubation period, the liver or other tissue cells burst open and release numerous merozoites into the bloodstream.
Shortly thereafter, certain of these blood borne merozoites invade red blood cells, where they enter the erythrocytic phase of the life cycle. Within the red blood cells, young plasmodia have a red nucleus and a ring-shaped, blue cytoplasm. The plasmodium divides into merozoites, which may break out of the red blood cell, enter other erythrocytes and repeat the multiplication process. This period lasts approximately 48 hours.
During this same 48 hour period of the erythrocytic cycle, male and female gametocytes are formed in the red blood cells. These gametocytes also burst out of the red blood cells along with the merozoites. It is during this period that the human host experiences the symptoms associated with malaria. The merozoites which burst forth from the red blood cells live for only a few hours in the bloodstream. The gametocytes live for several days or more in the host""s bloodstream.
The gametocytes are capable of mating only in the mosquito. Thus, in order for Plasmdium falciparium to produce sporozoites for infecting a second human host, a mosquito must first bite a human host carrying gametocytes. These gametocytes mature into macrogametes, mate in the mosquito""s stomach and produce a zygote. The zygote (ookinete) is active and moves through the stomach or the midgut wall. Under the lining of the gut, the ookinete becomes rounded and forms a cyst called an oocyst, in which hundreds of sporozoites develop. Sporozoites thereafter invade the entire mosquito and many of them enter the salivary glands where they are in a favorable position to infect the next host when the mosquito feeds on its blood. The life cycle thereafter simply repeats itself in another human host.
During the life cycle of Plasmdium falciparium, inhibition of invasion of the erythrocyte by the merozoite may be a key to developing an effective vaccine for malaria. Once the parasite has gained entry into the red cell, exposure to the immune system is gone.
In the past, live vaccinia virus was used as a vaccine to eradicate smallpox successfully, and a recombinant vaccinia virus expressing viral antigens has been shown to induce a strong antibody response in immunized animals, conferring protection against disease (Arita, I., Nature, 1979, 279, 293-298). Furthermore, it has been shown in animal models that co-presentation of potential immunogens with highly immunogenic vaccinia virus proteins can elicit a strong immune response against that specific immunogen (Moss and Flexner, Annals of the New York Academy of Sciences, 86-103; Mackett and Smith, J. Gen. Virol., 1986, 67, 2067-2082; Houard, et al., J. Gen. Virol., 1995, 76, 421-423; Fujii, et al., J. Gen. Virol., 1994, 75, 1339-1344; Rodrigues, et al. J. Immunol., 1994, 153, 4636-4648). Therefore, the utilization of live recombinant vaccinia virus as a vaccine might overcome many problems of antigen expression and delivery presently encountered in the preparation of recombinant proteins in E. coli, yeast or insect expression systems. A panel of transfer vectors have been constructed that allow insertion of foreign genes into several sites within the 180 kb vaccinia virus genome (Earl and Moss, Current Protocols in Molecular Biology, 1993, 16.17.1-16.17.16). Also, it has been reported that  greater than 25 kb of foreign DNA can be inserted into the vaccinia virus genome (Smith and Moss, Gene, 1983, 25, 21-28). The correct processing (Chakrabarta, et al., Nature, 1986, 320, 535-537) and the appropriate post-translational modification (Hu, et al., Nature, 1986, 320, 537-540; Ball, et al., Proc. Natl. Acad. Sci. USA, 1986, 83, 246-250; de la Salle, et al., Nature, 1985, 316, 268-270), transport and secretion (Ball, et al., Proc. Natl. Acad. Scienc. USA, 1986, 83, 246-250 and Langford, et al., Mol. Cell. Biol. 1986, 6, 3191-3199) are dictated by the primary structure of the expressed protein. In addition, a recombinant vaccinia virus vaccine has the advantage of being relatively inexpensive and easily stored, transported and delivered, features which are particularly important in the developing countries where malaria is most prevalent.
Proteins on the surface of merozoites are good targets for an immune response and are good malaria vaccine candidates because merozoites are the only stage in the asexual blood cycle in which the parasite is exposed to the immune system. The 190 kD glycoprotein of Plasmodium falciparum, precursor to major merozoite surface antigen1 (MSA1), which is synthesized during schizogony, is considered a promising candidate for a blood-stage malaria vaccine (Blackman, et al., Mol. Biochem. Parasitol., 1991, 49, 29-34; Perrin, et al., J. Exp. Med., 1984, 160, 441-451; Siddiqui, et al., Proc. Natl. Acad. Sci. USA, 1987, 84, 3014-3018; Perrin, et al., Immunol. Rev., 1982, 61, 245-269). The high-molecular weight precursor is processed into 88 kD, 30 kD, 38 kD and 42 kD fragments which remain as complexes on the merozoite surface (Holder, et al., Parasitology, 1987, 94, 199-208; McBride and Heidrich, Mol. Biochem. Parsitol., 1987, 23, 71-84). The complex is released from the membrane by cleavage of the 42 kD anchor fragment, and a 19 kD carboxyl-terminal fragment remains on the merozoite membrane and is carried into the invaded erythrocytes (Blackman, et al., supra; Blackman, et al., Mol. Biochem. Parasitol., 1991, 49, 35-44). The complete MSA1 of unprocessed P. falciparum has been used to provide partial or complete protection against challenge infection (Blackman, et al., Mol. Biochem. Parasitol., 1991, 49, 29-34; Perrin, et al., J. Exp. Med., 1984, 160, 441-451; Siddiqui, et al., Proc. Natl. Acad. Sci. USA, 1987, 84, 3014-3018), and it is highly immunogenic in humans (Perrin, et al., Immunol. Rev., 1982, 61, 245-269). Rabbit antibody against the C-terminal processing fragment of MSA1, as expressed in baculovirus, strongly inhibits parasite growth in vitro. These antibodies were able to inhibit homologous and heterologous parasites with similar degrees of efficiency (Hui, et al., Infect. Imm., 1993, 61, 3403-3411).
In prior work at Georgetown University, a series of monoclonal antibodies (mAbs) directed against glycophorin A, the putative erythrocyte receptor for P. falciparum were prepared. One of these mAbs, designated 2B10 is capable of blocking the binding of MSA1 to human erythrocytes and inhibiting the invasion of P. falciparum merozoites into human erythrocytes (Su, et al. Infect. Imm., 1993, 151, 2309). The anti-idiotype antibody of 2B10 recognized the C-terminal (1047-1640aa) region of MSA1 in a western blot (Su, et al., J. Immunol., 1995) and appears to recognize the same site on glycophorin A as the merozoite.
The present invention relates to a malaria vaccine comprising an expression vector, preferably, a vaccinia virus system which expresses a protein corresponding substantially to a specific domain of the major merozoite surface antigen 1 (MSA1) of Plasmodium falciparum or an immugenic peptide portion thereof.
In this preferred vaccinia virus system, the DNA coding for the MSA1 protein domain is expressed by the vaccinia virus after administration to a patient. The MSA1 protein or sub-fragment which is then expressed in the patient raises a humoral and/or cell-mediated response to the merozoite malaria antigen, which response provides the effect of protecting the vaccinated patient from a subsequent malaria infection. In preferred embodiments according to the present invention, the vaccinia virus system continues to express antigen in the patient for a period of days, months or even years, thus reinforcing the immunogenic response of the patient to the expressed antigen.
The MSA1 peptide antigen or immunogenic peptide portion thereof which is expressed by the expression vector vaccinia virus may also comprise a signal peptide and/or an anchor peptide sequence. It has been found that the addition of a signal and/or anchor peptide to the expressed MSA1 antigen in vaccines according to the present invention unexpectedly enhances the immunogenicity to the patient of the MSA1 protein of Plasmodium falciparum. It is an unexpected result that the inclusion of a signal and/or anchor protein with MSA1 can be expressed by a vaccinia virus system according to the present invention and the expressed peptide will produce a significantly greater immunogenic response than the MSA1 peptide alone or in combination with an adjuvant. It is also an unexpected result that the inclusion of a signal and anchor sequence in the MSA1 peptide sequence expressed by the vaccinia virus will produce an immunogenic response which may be as much as 100 fold greater than the immnogenic response which is produced by the MSA1 peptide which does not contain a signal or anchor peptide sequence.
Methods of inducing an immunogenic response in a patient are also contemplated by the present invention. In this method, a patient is administered an amount of a vaccinia virus capable of expressing the MSA1 peptide of Plasmodium falciparum such that the patient develops an immunogenic response to the expressed peptide. The immunogenic response generated preferably will be xe2x80x9csubstantially protectivexe2x80x9d, i.e., will protect the patient from some of the more severe symptoms and physiological states of the malaria disease, including the death of the patient from malaria.
The present invention also relates to an immunogenic dosage form as a vaccine, for inducing an immunogenic response to the merozoite stage in the life cycle of Plasmodium falciparum. Methods of vaccinating a patient against a malaria infection are also contemplated by the present invention. In this method, a patient is vaccinated against a Plasmodium falciparum infection by administering an immunogenic response producing effective amount of a vaccinia virus capable of expressing the MSA1 peptide or an immunogenic peptide portion thereof of Plasmodium falciparum in the patient.
The present invention also relates to chimeric proteins or peptides comprising the peptide sequence corresponding to the major merozoite surface antigen 1 (MSA1) of Plasmodium falciparum or an immugenic peptide portion thereof in combination with a signal sequence and/or anchor sequence, more preferably both a signal sequence and an anchor sequence.